Despite the recent completion of the human genome project, an ostensibly more difficult post-genomic challenge will be the functional annotation of all human genes and integration of this information into an operational cell-based model. Unfortunately, this is at present challenging, primarily due to the absence of suitable toolsets to rapidly delineate gene function en masse. RNA interference (RNAi) has proven to be an extremely potent and versatile tool to specifically reduce expression of targeted genes, allowing for loss-of- function genetic screens in mammalian cells. Despite these successes, high-throughput (HT) RNAi screening is technically challenging and significant limitations in the technology exist. To address these issues, and to expand on previous program funding, we have developed a novel experimental platform to construct functionally validated (FV) shRNA libraries. Under Phase I funding, we initially propose to develop a FV human shRNA library in biosafe lentiviral vectors targeting 5000 pharmaceutically tractable gene families that are amenable to small molecule drug discovery research. In collaboration with our biology consultants at the Fred Hutchinson Cancer Research Center (FHCRC) and the Scripps Research Institute (TSRI), we will subsequently validate this library in a simple cell culture-based model, to identify genes responsible for the growth limiting actions of TGF-[unreadable] in cancer cells. In Phase II, we will extend this program to include the development of genome-wide functionally validated human and mouse siRNA libraries. We propose to use our novel HT RNAi resource to delineate the processes which underlie deregulated proliferation in cancer cells, and to apply this knowledge into translational research programs. For example, we will use our newly developed FV siRNA library technology to identify synthetic lethal interactions in cancer cells. Genetic screens with FV siRNA libraries have the potential to greatly simplify gene manipulation and discovery for many biomedical applications, such as validation of gene function, probing interactions between genes, and the establishment of animal models. Moreover, we envisage a major impact on the molecular dissection of human disease mechanisms. For example, these reagents harbor considerable promise to identify new targets for therapeutic intervention, and the development of increasingly relevant paradigms for drug discovery. As a result, we foresee that these toolsets will significantly improve the efficiency, economy and ease of performing HT RNAi screens, and will provide basic researchers with preferred, cost-effective alternatives to existing commercially available reagents. The ultimate goal of the proposed project is to develop and make commercially available a new powerful research tool: a set of functionally validated genome-wide human and mouse lentiviral siRNA libraries designed for high-throughput discovery of novel drug targets. We propose to apply this powerful technology to identify novel anti- cancer drug targets. The developed technologies will significantly improve the efficiency of translational researches related to molecular dissection of diverse human disease mechanisms, development of new pharmaceuticals and therefore, have major implications for improving drug discovery research. [unreadable] [unreadable] [unreadable]